The use of PCR based methods in DNA fingerprinting has rapidly expanded the range of applications of DNA typing and genetic analysis in as widely diverse fields as microbial typing, plant and animal breeding and human genetic testing. These methods detect minor variations in the genetic material, termed DNA polymorphisms. The major limitation of current DNA typing technologies stems from the lack of DNA polymorphism in the genetic material under study. In general, DNA polymorphism results from random mutations, nucleotide changes or insertions and deletions, which have accumulated in the genetic material of each biological species. In certain species the frequency of random mutations appear to be high, while in other species such mutations occur rarely. As a consequence, DNA fingerprinting methods detecting random mutations will be highly informative in the former but not in the latter, thus severely limiting the use of DNA typing in the latter class of biological species. To overcome this limitation, DNA typing methods have been developed which target DNA segments or DNA sequences which exhibit a much higher degree of variability. One such type of hypervariable DNA sequences are the so called simple sequence repeats. These are DNA sequences composed of tandemly repeated sequences of one, two, three or four nucleotides. Occasionally repeat units of more than four nucleotides are observed. Such sequences generally exhibit a variation in the number of tandemly repeated units in genetic material and it is generally believed that this variation in repeat number arises from a high error rate during DNA replication. Since each repeat number of a simple sequence repeat constitutes a different allelic form, DNA markers based on simple sequence repeats are the most informative marker type currently available. The limitations of using simple sequence repeat DNA markers are twofold: (a) the development of these markers is extremely laborious and time consuming and needs to be repeated for each biological species and (b) the detection of these markers is limited to single locus assay systems in which the DNA markers are individually identified in separate PCR reactions.
In view of these limitations there is a strong need for DNA typing methods which would allow simple sequence repeat markers to be detected and isolated more efficiently, so as to broaden the range of applications of simple sequence repeat based marker systems.
It is the objective of the present invention to provide an efficient and generally applicable DNA fingerprinting method which obviates the heed for the laborious step in simple sequence repeat marker isolation and which provides a simple method for detecting a large number of simple sequence repeat markers in single multilocus assays.
The main problem in identifying simple sequence repeats in the genomes of biological species is that such sequences in general occur very infrequently, and hence very rarely appear in random DNA fingerprints. In the present invention we have devised a method for selectively amplifying simple sequence repeats in DNA and which can be displayed in DNA fingerprints. This method is based on an earlier invention in which a DNA fingerprinting method was developed to selectively amplify restriction fragments (EP0534858). In essence, the method for selective restriction fragment amplification as described in European Patent Application EP0534858 consists of digesting genomic DNA with restriction enzymes, ligating synthetic oligonucleotide adaptors to the ends, using "selective PCR primers" to amplify a subset of the restriction fragments, and fractionating the amplified fragments on an appropriate gel system. The selective principle resides in the design of the selective PCR primers. In general, these primers are composed of a sequence which matches the common sequences at the ends of the restriction fragments and a variable number of random nucleotides referred to as selective nucleotides, added to the 3'end of the common sequence. These selective nucleotides will ensure that only those restriction fragments exhibiting a matching sequence will be amplified. Since the 3'nucleotides must match perfectly in order for the PCR primers to efficiently amplify their target DNA fragment, this selective principle exhibits a very high degree of fidelity. This ensures that only those fragments having a perfect match to the selective nucleotides used will be amplified. Furthermore it has to be realized that the selection is applied at both ends simultaneously since both DNA strands need to be copied in order to achieve an exponential amplification in the PCR reaction. Extensive research has shown that the selective restriction fragment amplification method can be used effectively on DNA from any biological species to yield highly reproducible and detailed DNA fingerprints.
The preferred procedure for the selective restriction fragment amplification uses a combination of two different restriction enzymes: one enzyme which serves the purpose of targeting rare sequences (a rare cutter restriction enzyme) and a second enzyme (a frequent cutter enzyme) which serves the purpose of reducing the size of the restriction fragments to a range of sizes which are amplified efficiently. By targeting rare sequences one basically reduces the complexity of the starting mixture of DNA fragments, and hence one is able to achieve a more reliable and accurate amplification.
The DNA fingerprinting method for selective restriction fragment amplification detects two types of DNA markers: (a) dominant markers based on point mutations and (b) codominant markers based on insertions or deletions. In different DNAs with a high percentage of sequence polymorphism the method for selective restriction fragment amplification will generate lots of dominant markers. These dominant markers are mono-allelic. In more closely related DNAS, marker bands will be much less frequent, and consequently more fingerprints will have to be run in order to obtain enough markers. Moreover, the frequency in which codominant markers are detected is much lower than dominant markers. The simple sequence repeats constitute a special type of codominant marker. These repetitive elements usually display a high degree of length polymorphism. Moreover, there are often multiple alleles of these markers, giving these a high Polymorphism Information Content (PIC).
The method of the present invention provides an efficient and generally applicable DNA fingerprinting method in which preferably codominant markers are generated.